2. RESULTS
2.3. Non-targeted microarray analysis
2.3.1. UGT76B1 expression negatively regulates SA-mediated response
UGT76B1 has been shown to antagonistically impact on SA and JA pathways. To further identify genes regulated by different UGT76B1 expression levels, differential gene expressions were compared in ugt76b1-1 and UGT76B1-OE-7 relative to the wild type, Col-0.
In ugt76b1-1 expression of a total of 769 genes was altered twofold or more (P ≤ 0.05) as compared to Col-0. Among 769 genes, 539 genes were upregulated and 230 genes were downregulated (Figure 10). According to MapMan more than 250 out of 769 genes were related to pathogen defense response, most of which were induced in ugt76b1-1 (Figure 12 B). In UGT76B1-OE-7 expressions of 233 genes were changed twofold or more (P ≤ 0.05) compared with mock-treated Col-0. Among 233 genes, 64 genes were induced, whereas 169 genes were suppressed (Figure 10). About 90 out of 233 genes were related to defense response, most of which were suppressed by the constitutive expression of UGT76B1 (Figure 12C). A total of 97 genes were oppositely regulated by loss-of-function and ectopic expression of UGT76B1; the vast majority, 94 genes, were upregulated in the ugt76b1 knockout and downregulated by the UGT76B1 overexpression (Figure 13A). Only 3 genes were induced by overexpression of UGT76B1 but suppressed by the loss-of-function of UGT76B1 (Figure 13C). Surprisingly, 21 genes were induced in both lines with different UGT76B1 expression; however zero suppressed in both instances (data now shown). This strongly suggests that UGT76B1 has a function mainly in suppressing a set of defense-responsive genes.
Different UGT76B1 expression has been shown to manipulate SA metabolism, elaborated by elevated free SA in ugt76b1-1, but decreased free SA in UGT76B1-OE-7 (von Saint Paul et al., 2011). To explore whether the 94 genes negatively correlated with UGT76B1 expression respond to SA or JA stimuli or to infection by P. syringae, their expression in response to virulent Pseudomonas syringae pv. tomato DC3000 infection of wild type (vs. mock), Pseudomonas syringae pv. maculicola infection of npr1-1 or sid2 (vs. infected Col-0) or after treatment of Col-0 with BTH, SA or MeJA was extracted from the public database Genevestigator (https://www.genevestigator.com/gv/plant.jsp) (Zimmermann et al., 2005).
Sixty six genes could match the corresponding probes, since different platforms and annotations had been employed in these and my studies. These 66 genes were divided into two classes, sorted by less than twofold change (Table 1) vs. a higher extent in response to SA treatment (Table 2). Very few genes were induced lower than twofold in response to SA treatment, among which the responsiveness of RLP28 and RLP43, belonging to receptor-like
proteins was less than twofold to any elicitation from Genevestigator (Table 1). In contrast, RLP28 and RLP43 were more than twofold induced in ugt76b1, but suppressed in UGT76B1 overexpression (Table 1).This indicated that RLP28 and RLP43 might only be specifically regulated by UGT76B1, but not responsive to treatment with SA and BTH as well as Pseudomonas infection. Most of the UGT76B1-dependent genes can be induced by Pseudomonas infection as well as by BTH and SA stimuli (Table 2). The induction of most of them showed SID2 and NPR1 dependence when infected with Pseudomonas syringae pv.
maculicola, as compared to Pseudomonas syringae pv. maculicola infection of Col-0.
Furthermore, about one half of these sixty six genes were repressed by MeJA (Table 1 and 2).
Thus, UGT76B1 had a major role in suppressing a set of SA-responsive genes, which could be regulated via SID2 and NPR1 (Table 1 and 2).
A
B
C
Figure 12. Analysis of biotic stress relevance on genes regulated by ILA and different UGT76B1 expression.
(A) Two hundred thirty six genes, induced or suppressed more than twofold by ILA, were analyzed.
(B) Seven hundred sixty nine genes, induced or suppressed more than twofold by loss-of-function of UGT76B1, were analyzed. (C) Two hundred thirty three genes, induced or suppressed more than twofold by constitutive overexpression of UGT76B1, were analyzed.
A B
C
Figure 13. Venn diagrams of transcriptome analysis on action of ILA and different UGT76B1 expression.
(A) Expression patterns were compared among ILA-induced, ugt76b1-induced and UGT76B1-suppressed gene lists. (B) Expression patterns were compared among ILA-UGT76B1-suppressed, ugt76b1-suppressed and UGT76B1-induced gene lists. (C) Expression patterns were compared among ILA-induced, ugt76b1-suppressed and UGT76B1-induced gene lists.
Table 1. Comparison of genes oppositely regulated in ugt76b1 knockout and UGT76B1 overexpression relative to Col-0 with microarray data sets published in Genevestigator.
Ninety four genes induced in ugt76b1 knockout but suppressed in UGT76B1 overexpression, however, were uploaded to Genevestigator. Among 94 genes, 66 matched the Affymetrix probes, which were classified into two groups according to the fold change in response to SA treatment. The first group of genes, which has been induced less than twofold in response to SA treatment, is shown here. Genes induced more than twofold (log2-transformed value ≥ 1) are indicated in “blue”, whereas genes suppressed more than twofold are highlighted in “red” (log2-transformed value ≤ -1).
1 From GEO accession # GSE 5520. Arabidopsis leaf samples were inoculated with Pseudomonas syringae pv.
tomato DC3000 for 24 h before harvesting.
2 From GEO accession # GSE9955. Whole Arabidopsis Col-0 plants were treated with the salicylic acid analog benzothiadiazole (BTH) for 24 h before harvesting.
3 From GEO accession #GSE34047. Leaf samples of Col-0 were sprayed with 1 mM salicylic acid and incubated for 6 h before collecting samples.
4 From GEO accession #GSE17464. Leaf disc samples were collected from five-week-old Ler plants grown under short day (8 h light / 16 h dark, 120 μmol s-1 m-2) conditions, sprayed with 10 µM MeJA, covered with plastic bags and kept for one hour.
5 and 6 From GEO accession # GSE 18978. Leaves of npr1, sid2 and Col-0 (control) in Arabidopsis were injected with Pseudomonas syringae pv. maculicola for 24 h before collecting samples.
7 and 8 Leaves of ugt76b1 knockout and UGT76B1 overexpression lines in Col-0 background were sprayed with water, compared with Col-0 sprayed with water via microarray.
9 The average fluorescence of three biological replicates water-treated Col-0 from 7 and 8 in microarray analysis.
The log2-transformed fluorescence of any sample in this assay is from 4 to 20.
Fold change (log2-transformed data)
AGI P. syringae 1 BTH2 SA3 MeJA4 npr1-15 sid26 ugt76b1-17 UGT76B1-OE-78
fluorescence
in WT9 Annotation (TAIR 10)
AT2G33080 -0.02 0.17 0.14 -0.15 -0.65 -0.30 3.78 -1.41 5.14 RLP28
AT3G28890 0.65 0.79 0.60 0.06 -0.08 -0.65 2.28 -2.51 7.39 RLP43
AT5G52740 -0.24 3.04 0.29 -0.07 0.02 -0.14 2.36 -1.17 4.76 Copper transport protein family
AT3G20960 0.36 0.09 0.54 -0.10 -1.10 -0.94 1.25 -1.17 6.99 CYP705A33A
AT1G20350 0.25 1.22 0.59 0.07 -0.22 0.23 3.00 -1.08 6.28 TIM17-1
AT2G21850 0.38 0.20 0.89 -0.41 -1.46 -1.29 1.73 -1.19 5.22 Cysteine/Histidine-rich C1 domain family protein
AT3G04720 -0.11 1.83 0.97 -4.30 -0.01 0.78 1.50 -1.04 11.77 PR-4
AT2G15390 1.65 1.22 0.97 -1.27 -0.79 -0.38 1.88 -1.15 7.00 FUT4
Table 2. Comparison of genes oppositely regulated in ugt76b1 knockout and UGT76B1 overexpression relative to Col-0 with microarray data sets published in Genevestigator.
Ninety four genes induced in ugt76b1 knockout but suppressed in UGT76B1 overexpression, however, were analyzed using Genevestigator for expression after the indicated treatments. Out of 94 genes 66 matched the Affymetrix probes, which were classified into two groups according to fold change in response to SA treatment. The second group of genes, which were induced more than twofold (log2 -transformed value ≥ 1) in response to SA treatment, was sorted according to the suppression by MeJA.
Genes induced more than twofold are indicated in “blue”, whereas genes suppressed more than twofold (log2-transformed value ≤ -1) are indicated in “red”. The description of experiments is the same as in Table 1.
Fold change (log2-transformed data) AGI P. syringae1 BTH2 SA3 MeJA4 npr1-15 sid26
ugt76b1-17
UGT76B1-OE-78
fluorescence
in WT9 Annotation (TAIR 10)
AT5G45380 0.30 2.71 2.01 -4.43 -1.06 -2.37 2.04 -1.60 8.36 DUR3, solute:sodium symporters AT1G51800 1.18 0.81 2.80 -4.14 -3.06 -3.14 2.83 -1.18 5.97 Leucine-rich repeat protein kinase family protein AT4G01700 1.77 3.17 3.31 -4.03 -0.61 -1.53 1.88 -1.08 9.90 Chitinase family protein AT1G10340 1.85 2.13 2.38 -3.94 -2.30 -1.90 3.63 -1.26 6.73 Ankyrin repeat family protein AT3G26210 1.26 3.93 3.27 -3.93 -0.82 -0.48 1.85 -2.02 9.48 CYP71B23 AT3G47480 2.49 3.19 2.13 -3.83 -0.71 -0.72 3.44 -1.89 9.76 Calcium-binding EF-hand family protein AT3G13950 2.06 2.31 1.46 -3.72 -1.09 -0.69 4.35 -1.03 4.16 unknown protein AT3G28540 1.87 2.77 2.91 -3.38 -1.91 -2.33 2.22 -1.22 10.85 P-loop containing nucleoside
triphosphate hydrolases superfamily protein AT3G51330 1.13 3.33 2.75 -3.28 -2.09 -2.13 2.15 -1.41 6.23 Eukaryotic aspartyl protease family protein
AT3G26220 0.66 2.37 1.02 -3.23 -1.10 -0.44 1.58 -1.09 8.93 CYP71B3
AT1G03850 1.37 5.63 4.37 -2.84 -2.79 -2.75 1.92 -1.03 9.43 Glutaredoxin family protein AT5G42830 0.39 1.92 1.68 -2.72 -2.21 -1.70 2.53 -1.75 6.69 HXXXD-type acyl-transferase family protein AT4G25110 0.74 1.76 2.68 -2.69 -2.08 -2.44 1.90 -3.23 9.27 AtMC2,metacaspase 2 AT5G67450 0.72 1.38 2.38 -2.55 -2.02 -1.95 1.39 -1.07 5.54 AZF1, ZF1, zinc-finger protein 1 AT1G35710 2.87 3.38 2.14 -2.40 -1.09 -1.71 2.36 -1.78 6.55 Protein kinase family protein
with leucine-rich repeat domain AT2G04450 1.68 4.23 2.82 -2.29 -1.75 -1.91 3.74 -2.08 7.91 NUDT6, nudix hydrolase homolog 6 AT1G30900 2.08 3.71 3.00 -2.23 -2.36 -2.50 2.52 -1.77 7.51 VACUOLAR SORTING RECEPTOR 6 AT4G23610 2.01 3.03 1.88 -2.17 -1.88 -1.76 1.84 -1.93 7.33 Late embryogenesis abundant (LEA)
hydroxyproline-rich glycoprotein family AT4G20110 2.10 2.74 2.92 -2.06 -1.11 -1.33 2.05 -2.28 9.22 VSR7, VACUOLAR SORTING RECEPTOR 7 AT5G20400 0.35 1.48 1.58 -1.79 -1.06 -1.15 1.30 -1.66 7.58 2-oxoglutarate (2OG) and Fe(II)-dependent
oxygenase superfamily protein AT2G47130 2.24 3.97 3.22 -1.71 -1.44 -1.60 1.89 -2.52 7.57 NAD(P)-binding Rossmann-fold superfamily protein AT1G13470 2.38 5.16 3.62 -1.41 -2.72 -2.67 2.57 -4.75 10.76 Protein of unknown function (DUF1262) AT5G59680 0.49 1.61 1.50 -1.29 -1.37 -1.35 1.61 -2.33 7.50 Leucine-rich repeat protein kinase family protein AT4G11890 2.32 4.46 3.34 -1.07 -1.01 -0.93 2.78 -1.61 6.08 Protein kinase superfamily protein AT1G73805 2.41 2.38 2.42 -1.04 -1.52 -1.94 2.33 -1.98 9.21 Calmodulin binding protein-like AT5G10760 0.97 4.88 1.90 -1.00 -1.78 -2.06 4.02 -2.45 9.31 Eukaryotic aspartyl protease family protein AT5G24210 1.12 1.76 3.10 -1.00 -1.34 -1.27 1.81 -1.07 11.78 alpha/beta-Hydrolases superfamily protein
AT1G34420 2.44 0.93 1.20 -0.98 -0.44 -0.30 2.10 -2.20 7.79 leucine-rich repeat transmembrane protein kinase family protein AT5G59670 1.64 3.36 2.82 0.03 -1.47 -1.94 1.21 -2.22 11.50 Leucine-rich repeat protein kinase family protein AT5G60900 3.21 2.52 2.73 -0.82 -0.49 -0.81 1.36 -2.65 10.85 RLK1, receptor-like protein kinase 1 AT2G32680 0.96 3.55 2.29 0.35 -0.77 -0.54 3.80 -1.61 6.41 RLP23, receptor like protein 23 AT3G25010 1.90 3.80 2.16 -0.29 -0.29 -0.93 4.04 -1.44 6.26 RLP41, receptor like protein 41
AT3G25020 0.82 1.34 1.90 -0.17 -0.93 -1.26 2.58 -1.04 7.34 RLP42, receptor like protein 42
AT4G23220 1.68 2.94 2.75 0.22 -0.84 -0.96 1.32 -1.20 9.52 CRK14
AT3G45860 0.43 3.94 1.60 0.40 -1.20 -1.92 2.92 -1.78 5.66 CRK4, cysteine-rich RLK (RECEPTOR-like protein kinase) 4 AT4G23150 2.22 3.13 2.70 0.29 -1.08 -0.91 4.62 -1.77 5.99 CRK7, cysteine-rich RLK
(RECEPTOR-like protein kinase) 7 AT5G45000 1.80 2.45 2.57 -0.63 -2.91 -3.24 1.24 -2.70 7.63 Disease resistance protein
(TIR-NBS-LRR class) family AT1G21250 2.28 3.51 2.89 0.05 -0.75 -1.10 1.56 -2.17 11.25 PRO25, WAK1, cell wall-associated kinase AT5G24530 2.01 4.61 2.85 -0.07 -1.71 -2.59 2.56 -3.04 11.35 DMR6, 2-oxoglutarate (2OG) and Fe(II)-dependent
oxygenase superfamily protein AT4G10500 2.49 4.83 3.55 0.20 -2.02 -2.71 6.38 -1.99 5.88 2-oxoglutarate (2OG) and Fe(II)-dependent
oxygenase superfamily protein AT5G05460 1.41 1.72 2.10 -0.64 -1.37 -1.69 1.18 -1.85 8.04 Glycosyl hydrolase family 85 AT3G50480 1.10 3.08 2.47 0.11 -1.14 -0.70 2.42 -1.69 8.98 HR4, homolog of RPW8 4 AT5G09290 1.31 0.57 1.17 -0.01 -1.82 -2.03 3.45 -1.24 5.28 Inositol monophosphatase family protein AT2G39210 1.17 1.79 2.85 -0.86 -1.27 -1.51 1.31 -1.33 10.07 Major facilitator superfamily protein AT2G26560 0.37 3.60 2.02 -0.36 -1.30 -0.74 1.96 -1.95 10.60 PLA IIA, PLA2A, phospholipase A 2A AT2G26440 2.12 3.87 3.27 0.23 -1.73 -2.68 1.50 -2.73 11.61 Plant invertase
pectin methylesterase inhibitor superfamily AT2G32160 0.53 1.70 1.43 0.34 -1.45 -1.49 1.10 -1.68 7.63 S-adenosyl-L-methionine-dependent
methyltransferases superfamily protein AT5G07770 0.83 0.68 1.10 -0.06 -1.29 -1.56 1.05 -1.01 8.39 Actin-binding FH2 protein AT4G03450 0.94 2.42 2.46 0.99 -1.34 -1.09 1.95 -2.68 8.08 Ankyrin repeat family protein AT1G24150 1.38 1.65 1.77 0.24 -1.01 -0.87 1.24 -1.05 7.69 FH4, formin homologue 4
AT5G22570 3.77 5.35 4.71 -0.51 -5.12 -5.24 2.82 -3.15 3.42 WRKY38
AT3G57260 2.91 5.10 1.64 0.13 0.14 -0.19 5.38 -2.04 9.81 BG2,PR2
AT3G57240 1.32 4.76 3.74 -0.43 -1.91 -1.72 2.29 -2.37 4.65 BG3, beta-1,3-glucanase 3 AT5G55450 1.62 5.02 2.60 0.00 -0.44 -0.34 2.09 -2.06 5.93 Bifunctional inhibitor/lipid-transfer protein AT2G34940 1.68 1.48 2.84 -0.30 -1.86 -1.92 1.30 -2.51 6.99 BP80-3;2, VSR3;2, VSR5,
VACUOLAR SORTING RECEPTOR 5 AT5G52760 3.63 4.64 2.08 0.05 -1.10 -1.15 3.11 -1.61 8.07 Copper transport protein family AT1G23840 1.29 2.13 2.12 -0.83 -1.39 -1.36 1.55 -1.33 9.45 unknown protein AT3G48640 2.34 2.20 2.18 -0.37 -1.81 -1.70 2.00 -1.00 5.39 unknown protein
2.3.2. Microarray analysis indicates a negative correlation of UGT76B1 expression and